An isocratic high performance liquid chromatography (HPLC) method has been developed for the determination of Bifidobacterium bifidum growth factors in human milk. The method involves the gradual addition of 3 volumes of ethanol to the milk sample, filtration, and analysis of the growth factors in the filtrate by HPLC. The HPLC system consisted of a carbohydrate analysis column, a water-acetonitrile (70 + 30) solvent system, a flow rate of 1.0 mL/min, and a refractive index detector. The method is simpler and requires less time than the present microbiological method. Moreover, it revealed for the first time the presence of 2 separable growth factors in all human milk samples tested. The HPLC method developed is sensitive and can be used to monitor the type and the amount of growth factors in mothers' milk during lactation.